U.S. patent application number 12/459309 was filed with the patent office on 2010-12-30 for chemically induced optical signals and dna sequencing.
Invention is credited to Jianquan Liu, Xing Su, Liming Wang, Kai Wu.
Application Number | 20100330553 12/459309 |
Document ID | / |
Family ID | 43381147 |
Filed Date | 2010-12-30 |
![](/patent/app/20100330553/US20100330553A1-20101230-D00000.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00001.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00002.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00003.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00004.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00005.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00006.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00007.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00008.png)
![](/patent/app/20100330553/US20100330553A1-20101230-D00009.png)
United States Patent
Application |
20100330553 |
Kind Code |
A1 |
Su; Xing ; et al. |
December 30, 2010 |
Chemically induced optical signals and DNA sequencing
Abstract
Methods for sequencing nucleic acids are presented. Sequencing
is accomplished through the chemical amplification of the products
of DNA synthesis and the detection of the chemically amplified
products. In embodiments of the invention, a substrate is provided
having a plurality of molecules of DNA to be sequenced attached and
a plurality of molecules capable of chelating pyrophosphate ions
attached, the DNA molecules to be sequenced are primed, and a next
complementary nucleotide is incorporated and excised a plurality of
times leading to the buildup of pyrophosphate ions locally around
the DNA molecule to be sequenced. Pyrophosphate ions are captured
by the substrate-attached chelators and optically detected to
determine the identity of the next complementary nucleic acid in
the DNA molecule to be sequenced.
Inventors: |
Su; Xing; (Cupertino,
CA) ; Wang; Liming; (Santa Clara, CA) ; Liu;
Jianquan; (Fremont, CA) ; Wu; Kai; (Mountain
View, CA) |
Correspondence
Address: |
INTEL CORPORATION;c/o CPA Global
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
43381147 |
Appl. No.: |
12/459309 |
Filed: |
June 30, 2009 |
Current U.S.
Class: |
435/6.15 ;
385/129; 435/287.2 |
Current CPC
Class: |
G02B 6/1221 20130101;
C12Q 1/6874 20130101; C12Q 1/6874 20130101; C12Q 2527/125 20130101;
C12Q 2565/607 20130101; C12Q 2563/107 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 385/129 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/00 20060101 C12M001/00; G02B 6/10 20060101
G02B006/10 |
Claims
1. A method for analyzing a nucleic acid comprising: providing a
substrate capable of functioning as a waveguide having a surface
wherein the surface comprises an attached nucleic acid molecule to
be sequenced and a plurality of attached molecules capable of
chelating a pyrophosphate ion, terminating a complementary nucleic
acid polymer hybridized to the nucleic acid molecule to be
sequenced with a nuclease resistant nucleotide, providing a
solution comprising a polymerase enzyme, an exonuclease enzyme, and
a nucleotide triphosphate, under conditions that allow a
complementary nucleotide triphosphate to be incorporated into the
complementary polymer by the polymerase enzyme and excised from the
growing complementary strand by the exonuclease enzyme a plurality
of times thereby producing a plurality of pyrophosphate ions, and
under conditions that allow the molecules capable of chelating a
pyrophosphate ion to chelate phyrophosphate ions, detecting the
presence of pyrophosphate ions that are the product of
complementary nucleotide incorporation and excision, wherein
detection occurs through the detection of an optical signal
indicative of the chelation of pyrophosphate ions by attached
molecules capable of chelating pyrophosphate ions, and determining
the identity of a base of the nucleic acid to be sequenced through
the detection of the incorporation of a complementary
nucleotide.
2. The method of claim 1 wherein the substrate is a planar
waveguide.
3. The method of claim 1 wherein the substrate is a planar
waveguide that is comprised of SiO.sub.2.
4. The method of claim 1 wherein the substrate is a planar
waveguide and the optical signal is the product of evanescent
excitation of surface-attached molecules.
5. The method of claim 1 wherein the substrate is a zero mode
optical waveguide device.
6. The method of claim 1 wherein the substrate is a plasmonic
optical waveguide device.
7. The method of claim 1 wherein phyrophosphate chelator comprises
a detectable label that is displaced when the pyrophosphate
chelator chelates pyrophosphate ion and the detection of
pyrophosphate ions occurs through the detection of the absence of
the detectable label.
8. The method of claim 7 wherein the detectable label is a
fluorescent label.
9. The method of claim 1 wherein the terminating, providing,
detecting, and determining are performed a plurality of times and
sequence information is determined for a section of the nucleic
acid molecule to be sequenced comprising a plurality of bases.
10. The method of claim 1 wherein the surface comprises a plurality
of attached nucleic acid molecules to be sequenced.
11. A method for analyzing a nucleic acid comprising: providing a
substrate capable of functioning as a waveguide having a surface
wherein the surface comprises an attached nucleic acid molecule to
be sequenced and a plurality of attached molecules capable of
chelating a pyrophosphate ion, terminating a complementary nucleic
acid polymer hybridized to the nucleic acid molecule to be
sequenced with a nuclease resistant nucleotide, providing a
solution comprising a polymerase enzyme, an exonuclease enzyme, and
a nucleotide triphosphate wherein the nucleotide triphosphate
comprises an optically detectable label attached to a phosphate
group, under conditions that allow a complementary nucleotide
triphosphate to be incorporated into the complementary polymer by
the polymerase enzyme and excised from the growing complementary
strand by the exonuclease enzyme a plurality of times thereby
producing a plurality of labeled pyrophosphate ions, and under
conditions that allow the molecules capable of chelating a
pyrophosphate ion to chelate phyrophosphate ions, detecting the
presence of labeled pyrophosphate ions that are the product of
complementary nucleotide incorporation and excision, wherein
detection occurs through the detection of an optical signal from
the label indicative of the chelation of pyrophosphate ions by
attached molecules capable of chelating pyrophosphate ions, and
determining the identity of a base of the nucleic acid to be
sequenced through the detection of the incorporation of a
complementary nucleotide.
12. The method of claim 11 wherein the substrate is a planar
waveguide.
13. The method of claim 11 wherein the substrate is a planar
waveguide that is comprised of SiO.sub.2.
14. The method of claim 11 wherein the substrate is a zero mode
optical waveguide device.
15. The method of claim 11 wherein the substrate is a plasmonic
optical waveguide device.
16. The method of claim 11 wherein the label is detectable through
the detection of its fluorescence emission.
17. The method of claim 11 wherein the solution comprises four
different nucleotide triphosphates, wherein each of the four
different nucleotide triphosphates comprises a different
distinguishable label, and wherein detecting includes detecting the
identity of the different distinguishable label and thereby
detecting the identity of the nucleotide that was incorporated into
the nucleic acid molecule to be sequenced.
18. The method of claim 11 wherein the terminating, providing, and
detecting are performed a plurality of times and sequence
information is determined for a section of the nucleic acid
molecule to be sequenced comprising a plurality of bases.
19. The method of claim 11 wherein the surface comprises a
plurality of attached nucleic acid molecules to be sequenced.
20. A device comprising: a substrate that is capable of functioning
as a waveguide having a surface wherein the surface comprises a
plurality of attachment sites for nucleic acid molecules to be
sequenced and a plurality of attached molecules capable of
chelating a pyrophosphate ion, an optical system comprising a light
source, a light source control, and an image detector, an
electronics system operably coupling a computer to the optical
system, and the computer capable of receiving, storing, and
processing data from the electronics system in order to assemble
the sequence of a nucleic acid molecule to be sequenced.
21. The device of claim 20 wherein the substrate surface
additionally comprises a patterned metal layer.
22. The device of claim 20 wherein the substrate is a planar
waveguide.
23. The device of claim 20 wherein the substrate is a planar
waveguide comprised of SiO.sub.2.
24. The device of claim 20 wherein the substrate is a zero mode
optical waveguide device.
25. The device of claim 20 wherein the substrate is a plasmonic
optical waveguide device.
26. The device of claim 20 also comprising a fluid delivery system,
wherein the fluid delivery system is comprised of a plurality or
reservoirs capable of containing a plurality of solutions and a
plurality of outlets from plurality of reservoirs capable of
delivering fluids to the surface of the substrate.
27. The device of claim 20 wherein the electronics system is
capable of causing a solution from a reservoir to be supplied to
the surface of the substrate and wherein the computer is capable of
directing the electronics system to supply a solution from a
reservoir to the surface of the substrate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to U.S. patent
application Ser. No. 11/226,696, entitled "Sensor Arrays and
Nucleic Acid Sequencing Applications," filed Sep. 13, 2005, now
pending, which is a continuation-in-part application that claims
the benefit of U.S. application Ser. No. 11/073,160, entitled
"Sensor Arrays and Nucleic Acid Sequencing Applications," filed
Mar. 4, 2005, and is also related to U.S. patent application Ser.
No. 12/319,168, entitled "Nucleic Acid Sequencing and Electronic
Detection," filed Dec. 31, 2008, now pending, the disclosures of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The embodiments of the present invention relate generally to
methods and devices for nucleic acid sequencing and the optical
detection of the products of nucleic acid sequencing reactions.
[0004] 2. Background Information
[0005] Genetic information in living organisms is contained in the
form of very long nucleic acid molecules such as deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA). Naturally occurring DNA and
RNA molecules are typically composed of repeating chemical building
blocks called nucleotides which are in turn made up of a sugar
(deoxyribose or ribose, respectively), phosphoric acid, and one of
four bases, adenine (A), cytosine (C), guanine (G), and thymine (T)
or uracil (U). The human genome, for example, contains
approximately three billion nucleotides of DNA sequence and an
estimated 20,000 to 25,000 genes. DNA sequence information can be
used to determine multiple characteristics of an individual as well
as the presence of and or suceptibility to many common diseases,
such as cancer, cystic fibrosis, and sickle cell anemia.
Determination of the entire three billion nucleotide sequence of
the human genome has provided a foundation for identifying the
genetic basis of such diseases. A determination of the sequence of
the human genome required years to accomplish. Sequencing the
genomes of individuals provides an opportunity to personalize
medical treatments. The need for nucleic acid sequence information
also exists in research, environmental protection, food safety,
biodefense, and clinical applications, such as for example,
pathogen detection (the detection of the presence or absence of
pathogens or their genetic varients).
[0006] Thus, because DNA sequencing is an important technology for
applications in bioscience, such as, for example, the analysis of
genetic information content for an organism, tools that allow for
faster and or more reliable sequence determination are valuable.
Applications such as, for example, population-based biodiversity
projects, disease detection, personalized medicine, prediction of
effectiveness of drugs, and genotyping using single-nucleotide
polymorphisms, stimulate the need for simple and robust methods for
sequencing short lengths of nucleic acids (such as, for example,
those containing 1-20 bases). Sequencing methods that provide
increased accuracy and or robustness, decreased need for analysis
sample, and or high throughput are valuable analytical and
biomedical tools.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 provides a simplified diagram of a method for the
parallel sequencing of nucleic acids employing chemical signal
amplification and optical detection of chemically amplified
sequencing reactions.
[0008] FIG. 2 shows an outline for a general nucleic acid
sequencing strategy using the chemical amplification of reaction
products and optical detection of amplified reaction products to
assemble sequence information.
[0009] FIGS. 3A and 3B diagram how a chemically-amplified localized
optical signal is generated during the sequencing reaction for a
surface-attached nucleic acid.
[0010] FIG. 4 diagrams multiplexed sequencing reaction for a
plurality of DNA molecules.
[0011] FIG. 5 provides an exemplary fluorescently labeled
deoxynucleotide triphosphate (dNTP).
[0012] FIG. 6 shows an exemplary diagram of a pyrophosphate
chelating molecule that can be attached to a surface.
[0013] FIG. 7 provides an exemplary planar waveguide structure
showing the direction of propagating light.
[0014] FIG. 8 provides a schematic diagram of a DNA sequencing
system.
[0015] FIG. 9 shows an exemplary synthesis scheme for a
surface-attached chelating molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Embodiments of the present invention provide devices and
methods for sequencing and detecting nucleic acids. Methods are
provided according to embodiments of the invention by which whole
genomes of organisms can be sequenced. In general, the types of
nucleic acids that can be sequenced include polymers of
deoxyribonucleotides (DNA) or ribonucleotides (RNA) and analogs
thereof that are linked together by a phosphodiester bond. A
polynucleotide can be a segment of a genome, a gene or a portion
thereof, a cDNA, or a synthetic polydeoxyribonucleic acid sequence.
A polynucleotide, including an oligonucleotide (for example, a
probe or a primer) can contain nucleoside or nucleotide analogs, or
a backbone bond other than a phosphodiester bond. In general, the
nucleotides comprising a polynucleotide are naturally occurring
deoxyribonucleotides, such as adenine, cytosine, guanine or thymine
linked to 2'-deoxyribose, or ribonucleotides such as adenine,
cytosine, guanine, or uracil linked to ribose. However, a
polynucleotide or oligonucleotide also can contain nucleotide
analogs, including non-naturally occurring synthetic nucleotides or
modified naturally occurring nucleotides.
[0017] The covalent bond linking the nucleotides of a
polynucleotide generally is a phosphodiester bond. However, the
covalent bond also can be any of a number of other types of bonds,
including a thiodiester bond, a phosphorothioate bond, a
peptide-like amide bond or any other bond known to those in the art
as useful for linking nucleotides to produce synthetic
polynucleotides. The incorporation of non-naturally occurring
nucleotide analogs or bonds linking the nucleotides or analogs can
be particularly useful where the polynucleotide is to be exposed to
an environment that can contain nucleolytic activity, since the
modified polynucleotides can be less susceptible to
degradation.
[0018] Virtually any naturally occurring nucleic acid may be
sequenced including, for example, chromosomal, mitochondrial or
chloroplast DNA or ribosomal, transfer, heterogeneous nuclear or
messenger RNA. RNA can be converted into more stable cDNA through
the use of a reverse transcription enzyme (reverse transcriptase).
Additionally, non-naturally occurring nucleic acids that are
susceptible to enzymatic synthesis and degredation may be used in
embodiments of the present invention.
[0019] Methods for preparing and isolating various forms of nucleic
acids are known. See for example, Berger and Kimmel, eds., Guide to
Molecular Cloning Techniques, Methods in Enzymology, Academic
Press, New York, N.Y. (1987); Sambrook, Fritsch and Maniatis, eds.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1989); and Ausbel, F. M., et al.,
eds., Current Protocols in Molecular Biology, Wiley and Sons, Inc.
(2007). Samples comprising RNA can be converted to DNA for
sequencing using a reverse transcriptase enzyme to synthesize a
complementary strand of DNA from the RNA molecule. Commercial kits
for preparing nucleic acids are available, such as, for example,
the SuperScript.TM. Double-Stranded cDNA Synthesis Kit from
Invitrogen.
[0020] Methods are provided for sequencing nucleic acids in which
amplification of the nucleic acid sample (i.e., increasing the
number of copies of the nucleic acid molecules in the sample)
optionally does not have to occur. As much as one third of the
error during the sequencing of a nucleic acid sample has been
reported to be due to errors introduced during the amplification of
the nucleic acid sample. By not amplifying the sample to be
sequenced, amplification-related errors can be avoided.
Additionally, avoiding amplifying a sample avoids the concentration
bias that can develop when a sample is amplified. The concentration
bias that occurs during amplification is a result of the selective
amplification advantage found for certain sequence populations,
such that some sequences are amplified preferentially to a greater
extent than other sequences. Because amplification-related errors
are reduced, the methods of the present invention are useful for
surveying for rare mutations among samples having a variety of
components (mixed background components).
[0021] FIG. 1 provides a depiction of a generalized sequencing
strategy according to embodiments of the invention. In FIG. 1, an
array of detection regions 100, such as, for example, a zero-mode
optical waveguide device, having reaction regions 110 and
immobilized DNA molecules 120 is shown. One DNA molecule to be
sequenced is immobilized per detection region 110 in this example.
Before sequencing a sample of DNA, overlapped DNA fragments are
immobilized randomly on the surface of a substrate so that
statistically one DNA molecule 120 occupies the reaction region 110
of a detection region 100. A sample of DNA can be fragmented into
smaller polymeric molecules using, for example, restriction enzymes
or mechanical forces (shearing). The immobilized nucleic acid is
primed with a primer 125 that is terminated with a nuclease
resistant base and nucleic acid synthesis and deconstruction
reactions are performed and amplified chemical products of the
synthesis reactions 130 are created in the detection regions 110.
The identified base position is then filled with a nuclease
resistant base, and the reaction is repeated to determine a
matching base for the next available position on the DNA strand to
be sequenced. In this example, the amplified chemical products 130
are detected optically and sequence data for the immobilized DNA
molecules is assembled. Reaction products in an array and their
corresponding positions and optical signals are recorded and
analyzed with a computer and software. Data from regions having no
immobilized nucleic acid sample or a plurality of immobilized
samples can be distinguished.
[0022] FIG. 2 provides an exemplary method for providing amplified
chemical signals and sequencing data for nucleic acid sequencing
reactions. In the method of FIG. 2, the chemical products resulting
from the incorporation of a complementary dNTP (deoxynucleotide
triphosphate) into a nucleic acid strand to be sequenced are
amplified through the repeated addition and excision of the next
complementary nucleotide. The DNA molecule to be sequenced is
primed with a primer that is terminated with an exonuclease
resistant nucleotide. In one embodiment, individual reactions are
performed using one of four dNTPs and a determination is made
regarding the next complementary nucleotide in the nucleic acid to
be sequenced. In general, a test reaction comprises a polymerase,
an exonuclease, and a deoxynucleoside triphosphatase (dATP
(deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),
dGMP (deoxyguanosine triphosphate), or dTMP (deoxythymidine
triphosphate), for example). A complementary nucleotide is
incorporated into the primed growing DNA molecule that is
terminated with a nuclease resistant base through the action of a
polymerase enzyme. Typical useful polymerase enzymes include DNA
polymerases, such as for example, E. coli DNA polymerase I and the
commercially available 9 N and Therminator DNA polymerases
(available from New England Biolabs, Inc., Beverly, Mass.). Thus,
for example, where there is a cytosine on the strand to be
sequenced, a guanine will be incorporated, where there is a
thymidine, an adenosine will be incorporated, and vice versa. If
the nucleoside triphosphate is incorporated into the growing strand
in the test reaction, then a pyrophosphate ion (a "pyrophosphate,"
"PPi," or P.sub.2O.sub.7.sup.-4) is released. In an amplification
reaction, an exonuclease is used to remove the incorporated
nucleoside monophosphate (NMP.sup.-2), allowing another
complementary nucleoside triphosphate to be incorporated and a
second PPi to be released. Repetition of these addition and
excision reactions provides amplification of reaction products.
Thus, a positive test reaction (i.e., the detection of chemically
amplified products) indicates that the base on the template DNA
strand to be sequenced immediately after the priming base (the 3'
base) of the primer strand is complementary to the test base (the
one of four dNTPs that was used in the synthesis and deconstruction
reaction). To sequence the next base on the template, the first
identified base on the primer strand is filled or replaced with a
nuclease-resistant nucleotide that then becomes the priming base
for the test reaction. Nuclease-resistant nucleotides can be
ribonucleotides or other modified nucleotides. A variety of
polymerases are available that can incorporate ribonucleotides or
modified nucleotides into DNA, such as for example, the
commercially available Therminator DNA polymerase (available from
New England Biolabs, Inc., Beverly, Mass.). See also, for example,
DeLucia, A. M., Grindley, N. D. F., Joyce, C. M., Nucleic Acids
Research, 31:14, 4129-4137 (2003); and Gao, G., Orlova, M.,
Georgiadis, M. M., Hendrickson, W. A., Goff, S. P., Proceedings of
the National Academy of Sciences, 94, 407-411 (1997). Exemplary
nuclease resistant bases include alpha-phosphorothioate
nucleotides, and exemplary nucleases that cannot digest these
resistant bases include exonuclease III. Reactions in which no
product is detected indicate that the test reaction provided a
nucleotide that was not complementary to the next base of the
nucleic acid to be sequenced.
[0023] FIGS. 3A and 3B diagram a sequencing reaction for a nucleic
acid molecule attached to a substrate. In FIG. 3A, a substrate 200
consists of a waveguide 205, a functional layer 210, a layer of
attached pyrophosphate (PPi) chelator 215, and a molecule of DNA to
be sequenced 220. The molecule of DNA to be sequenced 220 and the
attached PPi chelator are attached to the functional layer 210
which facilitates light distribution or molecular attachment. In
this example, the functional layer 210 is a lower index of
refraction layer and can be considered to include any linker
molecules, the nucleic acid molecules and chelating molecules, and
liquid that may extend up to about 100 nm away from the waveguide
core. The functional layer 210 has a refractive index that is less
than the refractive index of the waveguide layer. Optionally, the
surface of the substrate comprises a patterned metal layer (not
shown) that facilitates other optical techniques through the
creation of surface plasmons or zero mode optical waveguide
phenomena. Typically, the optional patterned metal layer is a thin
layer having a thickness of between 1 nm and 100 nm and is
comprised of a metal such as for example, silver, gold or copper.
The surface-attached molecule of DNA 220 is primed with a short
hybridized complementary strand of DNA 225, also known as a priming
molecule or primer. The primer molecule 225 is terminated with a
nuclease resistant nucleotide. A solution is provided to the
surface of the substrate 200, comprising one or more types of
fluorescently labeled dNTPs (labeled F-PPP-N in FIG. 3A), a DNA
polymerase enzyme, and an exonuclease. The fluorescently labeled
dNTPs can be a single type of dNTP or a solution containing
multiple dNTPs, such as fluorescently labeled dATP (deoxyadenosine
triphosphate), dCTP (deoxycytidine triphosphate), dGMP
(deoxyguanosine triphosphate), and dTMP (deoxythymidine
triphosphate). For a solution of multiple types of dNTPs, each type
of dNTP can be labeled with a different fluorescent label wherein
the different fluorescent labels can be distinguished from each
other spectroscopically. The incorporation and excision reactions
of a next complementary nucleotide are allowed to proceed to create
a build-up of reaction products including the fluorescently-labeled
PPi molecules. Fluorescently labeled PPi molecules are captured by
the surface-attached PPi chelator molecules 215. A region 230
around the surface-attached DNA molecule 220 develops in which PPi
chelator molecules 215 have bound fluorescently-labeled PPi.
Evanescently-generated fluorescent signals 235 can be detected
using the waveguide to supply excitory radiation and a detector
positioned above (not shown) the substrate 200 to receive and
detect fluorescent radiation from the excited label. When the
incorporation of a nucleotide has been detected or at the end of
the test reaction, the substrate 200 is washed of the reactants,
including the fluorescently-labeled PPi that is bound to the
surface-attached chelators 215 and a next complementary
nuclease-resistant nucleotide is incorporated into the priming
strand 225, if the identity of the next complementary nucleotide is
known. The above reactions are repeated for the next complementary
nucleotide(s) to be determined and the sequence of the
surface-attached DNA strand is assembled.
[0024] In FIG. 3B, a substrate 300 consists of a waveguide 305, a
functional layer 310, and a layer of attached fluorogenic
pyrophosphate (PPi) chelator 315, and a molecule of DNA to be
sequenced 320. The molecule of DNA to be sequenced 320 and the
attached PPi chelator are attached to the functional layer 310. The
molecule of DNA to be sequenced 320 and the attached PPi chelator
are attached to the functional layer 310 which facilitates light
distribution or molecular attachment. The functional layer 310 has
a refractive index that is less than the refractive index of the
waveguide layer. In this example, the functional layer 310 is a
lower index of refraction layer and can be considered to include
any linker molecules, the nucleic acid molecules and chelating
molecules, and liquid that may extend up to about 100 nm away from
the waveguide core. Optionally, the surface of the substrate
additionally comprises a patterned metal layer (not shown) that
facilitates other optical waveguide techniques through the creation
of surface plasmons or zero mode optical phenomena. Typically, the
optional patterned metal layer is a thin layer having a thickness
of between 1 nm and 100 nm and is comprised of a metal such as for
example, silver, gold or copper. The surface-attached molecule of
DNA 320 is primed with a short complementary strand of DNA 325. The
primer molecule 325 is terminated with a nuclease resistant
nucleotide. A solution is provided to the surface of the substrate
300, comprising dNTPs (labeled PPP-N in FIG. 3B), a DNA polymerase
enzyme, and an exonuclease. The dNTPs can be a single type of dNTP
or a solution containing multiple dNTPs, such as dATP
(deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),
dGMP (deoxyguanosine triphosphate), and or dTMP (deoxythymidine
triphosphate). The incorporation and excision reactions of a next
complementary nucleotide are allowed to proceed to create a
build-up of reaction products including a plurality of PPi
molecules. The PPi molecules are captured by the surface-attached
fluorogenic PPi chelator molecules 315. A region 330 around the
surface-attached DNA molecule 320 develops in which PPi chelator
molecules 315 have bound PPi. Evanescently-generated fluorescent
signals 335 (for the cases in which evanescence is used to excite
fluorescent labels) are detected using the waveguide to supply
excitory radiation and a detector positioned above (not shown) the
substrate 300 to receive and detect fluorescent radiation from the
surface-attached fluorogenic chelator 315. In the alternative, when
binding of PPi by the chelator releases a fluorescent dye, a region
of no fluorescence indicates the production of reaction products
and the incorporation of a complementary nucleotide. When the
incorporation of a nucleotide has been detected or at the end of
the test reaction, the substrate 300 is washed of the reactants,
including the PPi that is bound to the surface-attached chelators
315 and a next complementary nuclease-resistant nucleotide is
incorporated into the priming strand 325, if the identity of the
next complementary nucleotide is known. The above reactions are
repeated for the next complementary nucleotide(s) to be determined
and the sequence of the surface-attached DNA strand is
assembled.
[0025] FIG. 4 provides a diagram of parallel sequencing reactions
for individual different DNA molecules. In FIG. 4, a substrate 400
comprises a waveguide 405 and individual DNA molecules 410 to be
sequenced (labeled in FIG. 4 "a" through "f"). The DNA molecules
410 are attached to the surface of substrate 400. The substrate 400
surface additionally comprises surface-attached PPi chelating
molecules (not shown). Sequencing reactions with chemical
amplification of reaction products are performed on the
surface-attached DNA molecules 410. The surface-attached DNA
molecules 410 are primed (primer not shown) and terminated with a
nuclease resistant nucleotide. A solution is provided to the
surface of the substrate 400, comprising four types of
fluorescently labeled dNTPs, a DNA polymerase enzyme, and an
exonuclease. The fluorescently labeled dNTPs are fluorescently
labeled dATP, dCTP, dGMP, and dTMP. In the example shown in FIG. 4,
each type of nucleotide is labeled with a different distinguishable
fluorescent label. The incorporation and excision reactions of a
next complementary nucleotide are allowed to proceed to create a
build-up of reaction products including a plurality of
fluorescently-labeled PPi molecules. Fluorescently labeled PPi
molecules are captured by the surface-attached PPi chelator
molecules (not shown). A region around the surface-attached DNA
molecule 410 develops in which PPi chelator molecules have bound
fluorescently-labeled PPi molecules. Evanescently-generated
fluorescent signals 415 can be detected using the waveguide to
supply excitory radiation and a detector positioned above (not
shown) the substrate 400 to receive and detect fluorescent
radiation from the excited labels. The different distinguishable
fluorescent labels are detected and distinguished (the labels
fluoresce at different wavelengths) to determine the identity of
the next complementary nucleotide. When the incorporation of a
nucleotide has been detected or at the end of the test reaction,
the substrate 400 is washed of the reactants, including the
fluorescently-labeled PPi that is bound to the surface-attached
chelators and a next complementary nuclease-resistant nucleotide is
incorporated into the priming strand (not shown), if the identity
of the next complementary nucleotide is known. The above reactions
are repeated (labeled "Cycle 2-Cycle n" in FIG. 4) for the next
complementary nucleotides to be determined and the sequence of the
surface-attached DNA strand is assembled.
[0026] If a nucleoside triphosphate is incorporated into the
growing strand in the test reaction, then a pyrophosphate (PPi) is
released. The pyrophosphate can be degraded into two inorganic
phosphates through ionic dissociation caused by water and catalyzed
by pyrophosphatase. In an amplification reaction, an exonuclease is
optionally used to remove the incorporated nucleoside
monophosphates (NMP.sup.-2), allowing another nucleoside
triphosphate to be incorporated and a PPi to be released.
Repetition of nucleotide incorporation and excision reactions
provides chemical amplification of inorganic phosphate
concentrations. Optionally, the nucleotide that is incorporated
into the growing polymer is labeled and a buildup of labels is
detected.
[0027] Nucleotides useful in the present invention include regular
deoxyribonucleoside triphosphates (dNTP) and fluorescent dye-tagged
dNTPs in which the fluorescent dye is attached to the
gamma-phosphate of the dNTP (fluor-dNTP). The dNTP can also be
fluorogenic, meaning that the intact fluor-dNTP is not fluorescent,
but when the fluor-dNTP is hydrolyzed creating fluor-PPi or just
fluor the dye's fluorescence becomes detectable. The phosphate
groups on the fluor-PPi molecule are removable, for example,
through the action of a phosphatase enzyme. The phosphatase enzyme
optionally is included in the reaction solution comprising dNTPs, a
DNA polymerase enzyme, and an exonuclease. In the situations in
which phosphatase and or pyrophosphatase are used, detection of
reaction products occurs in solution. In this case, metal nanogaps
are a suitable structure for the signal generation and detection
because the gaps can confine the signals to local areas within
given periods of time. Pyrophosphate (PPi) or fluor-PPi is the
byproduct of DNA polymerase reactions that incorporate
complementary nucleotides into hybridized growing DNA molecules and
PPi or fluor-PPi can be specifically captured by a chelating
molecule. Nuclease resistant nucleotides include, for example,
alpha-thiotriphosphate, alpha-methyltriphosphate, and
alpha-boranophosphate nucleotides.
[0028] FIG. 5 provides the structure of an exemplary
fluorescently-labeled nucleotide. In this example, the gamma
phosphate of a dGTP has been labeled with ATTO Rho6G (rhodamine
6G). The labeled nucleotide ATTO Rho6G-dGTP can be incorporated
into a growing DNA strand using, for example, 9 N and therminator
DNA polymerases. Additional examples of fluorescent labels that can
be attached to the gamma phosphate of a dNTP include, for example,
cyanine dyes such as Cy3 and Cy5, rhodamine derivatives MR200-1 and
JA169, oxazine derivative JA242 (see Lieberwirth, U. et al.,
Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by
Diode Laser-based Time-reolved Fluorescence Detection, Anal. Chem.,
70:4771-4779 (1998) and Rosenblum, B. B., et al., New Dye-labeled
Terminators for Improved DNA Sequencing Patterns, Nucleic Acids
Research, 25:4500-4504 (1997)), and fluorescein derivatives (see
Ju, J., et al., Fluorescence Energy Transfer Dye-labeled Primers
for DNA Sequencing and Analysis, Proc. Natl. Acad. Sci, USA,
92:4347-4351 (1995)). In general, a large number of fluorescent
dyes exist in the literature and are available for purchase from
commercial sources. Further, nucleotides include nucleotide analogs
and labeled nucleotide analogs, including methylated nucleotides,
non-naturally occurring synthetic nucleotides, and or modified
naturally occurring nucleotides.
[0029] In general, pyrophosphate chelators can be fluorescent after
binding a PPi molecule or a fluorescent dye can be released when
the chelator binds a PPi. In the case in which a fluorescent dye is
released through the binding of a PPi molecule with a
surface-attached chelating molecule, a region of no fluorescent
emission during a sequencing reaction of a surface-attached DNA
molecule indicates PPi production and the incorporation of a
complementary nucleotide. An exemplary chelating molecule that can
be attached to a surface is shown in FIG. 6. In FIG. 6, X
represents an surface attachment site for the chelating molecule
and can be a group such as, for example, a --NH.sub.2 group, an
--OH group, a halogen, a thiol, a carboxyl group, an aldehyde, or
an --NH--NH.sub.3 group. The "L" in FIG. 6 represents a spacer with
functional groups or a linker group and can be a group, such as for
example, a polyethyle glycol (PEG), polyphosphate
((PO.sub.4).sub.n), a structure such as (--C--).sub.n which is from
1 to 100 atoms in length and can contain functional groups such as
amine, hydroxyl, epoxy, aldehyde, carboxyl, and or thiol. The PPi
chelating portion of the molecule (the ligand portion) is
represented by the semicircle having an attached Y, in which Y is a
dye or cofactor for the chelator such as metal ions, such as, for
example, Zn.sup.2+, Cu.sup.2+, and or Fe.sup.3+. See FIG. 9 for an
exemplary chelator. A survey of molecules that are specific PPi
chelators can be found in Kim, S. K., et al, "Chemosensors for
Pyrophosphate," Acc. Chem. Res., 42: 23 (2008).
[0030] In general, the substrate is a planar waveguide (or slab
waveguide), a zero mode optical waveguide device, or a plasmonic
waveguide device. Planar waveguides typically have a rectangular
geometry and consist of at least three layers of material having
different dielectric constants. Light is confined to the middle
layer by total internal reflection which occurs when the dielectric
index of the middle layer is larger than that of the surrounding
layers. In the planar waveguide, light is injected into the side of
the waveguide as shown, for example, in FIG. 7. The critical angle
for light injection depends on the index of refraction of the
materials, which may vary depending on the wavelength of the light.
Such propagation will result in a guided mode only at a discrete
set of angles where the reflected planewave does not destructively
interfere with itself. In the case of the planar waveguide, the
substrate is constructed so that the molecular attachment area is
evanescent and fluorescent dye molecules are excited only when they
are in the proximity of the evanescent area. In this case, the
functional layer is the organic structure between the waveguide
layers and the DNA and chelator molecules (such as, for example, an
organic linker or an organic linker and a silanation layer). It
should have a refractive index that is lower than the middle
waveguide material. Pyrophosphate or fluor-PPi produce detectable
optical signals when the molecules are concentrated locally around
the surface-attached DNA molecules during polymerase reactions. The
reactions and optical signal recording are repeated in parallel for
a set of immobilized DNA molecules and DNA sequence information is
collected based on optical signal positions, timing, and wavelength
of fluorescent emission. In general a waveguide is a physical
structure that guides electromagnetic waves. Planar waveguides, for
example, can be comprised of SiO.sub.2, having surrounding layers
with a different (lower) refractive index and can be formed, for
example, from ion-exchange processes. See, for example, Haquin, H.,
et al., Recent Developments in Ion-exchanged Fluoride Glass Planar
Waveguides, J. Non-Crystalline Solids, 236-7:460-463 (2003) and
Navarro, A. G., Silica Waveguide Design and Fabrication using
Integrated Optics: A Link to Optical VLSI Photonics Integration for
Semiconductor Technology, 22.sup.nd Annual Microelectronic
Engineering Conference, 64-70 (May 2004). FIG. 7 provides an
exemplary planar waveguide structure 505. In FIG. 7, a planar
waveguide 510 is bounded on one side with a low refractive index
material 515 and a low refractive index functional layer 520.
Chelating molecules 525 and nucleic acids to be sequenced 530 are
attached to the functional layer 520. An arrow 535 shows the
direction of propagating light. Evanescence from the propagating
light within the waveguide 510 is created in the region of the
chelators 525. The evanescence is used to detect the chelation of
reaction products from DNA sequencing reactions.
[0031] In general, zero mode optical waveguide devices are
subwavelength optical nanostructures. To form a zero mode optical
waveguide device, a transparent substrate or a substrate having a
transparent surface layer is coated with a thin patterned metal
layer forming the optical nanostructures. The optical
nanostructures are sub-wavelength-sized holes in the metal layer.
Two different resonance sizes can be used to design the structures:
one is for excitation resonance and another is for emission
resonance. Typically, the hole is round in shape and its diameter
is less than one half of the wavelength of the light. the optional
patterned metal layer is a thin layer having a thickness of between
1 nm and 100 nm and is comprised of a metal such as for example,
silver, gold or copper. See, for example, Samiee, K. T., et al.,
Zero Mode Waveguides for Single-molecule Spectroscopy on Lipid
Membranes, Biophys. J., 90:3288-3299 (2006) and Levene, M. J., et
al., Zero-mode Waveguides for Single-molecule Analysis at High
Concentrations, Science, 299:682-686 (2003). DNA to be sequenced is
located within the hole through statistically random attachment
schemes. In these embodiments, some holes will have one DNA to be
sequenced immobilized, no DNA immobilized, or two or more nucleic
acids to be sequenced immobilized. Holes having no nucleic acid or
two or more nucleic acids immobilized are ignored. The transparent
material is a material such as, for example, SiO.sub.2, silicon
nitride, or a glass or quartz layer.
[0032] Further, the substrate can be a plasmonic waveguide device.
In a plasmonic waveguide device, a thin patterned metal layer
confines and guides light. The light is emitted at the edges of the
metal layer. The substrate on which the metal layer is patterned
does not need to be transparent and a variety of materials are
possible. For example, the substrate can be silicon, silicon
dioxide, glass, or a polymer. In these plasmonic waveguide
embodiments the thickness and type of metal layer are important to
the operation of the device. Typically the metal layer has holes
that have a linear or rectangular shape in which the longest
dimension of the hole is optionally larger than the wavelength of
light used to probe the DNA sequencing reaction. The nucleic acid
to be sequenced can be attached either at the edge of the metal
surrounding the hole or in the region of the waveguide surface
having no metal. In one embodiment, the chelators and nucleic acid
molecules to be sequenced are attached in the holes. One nucleic
acid molecule to be sequenced is attached in one hole (attached so
that statistically one nucleic acid molecule occupies one hole.)
Metals that are useful include copper, silver, gold and aluminum,
for example. Exemplary plasmonic waveguide devices include those
described in the following reference: Jun, Y. C., et al., Broadband
Enhancement of Light Emission in Silicon Slot Waveguides, Optics
Express, 17:7479-7490 (2009).
[0033] Affinity agents (PPi chelators) and DNA molecules to be
sequenced are co-immobilized on optical substrates (such as
waveguides). For example, the waveguide surface is functionalized
with one of or combination of amine, aldehye, epxoy, thiol, groups,
and DNA can be functionalized with amine (for surface bearing
carboxy, epoxy, and or aldehyde functional groups) and carboxyl
(for surface bearing amine groups), thiol (for surface of gold)
Various conjugation chemistries are available to join the
functional groups (for example, EDC for amine-carboxyl). The
concentration of DNA molecules can be controlled in several ways:
by limiting the density of surface functional groups or by limiting
the quantity of DNA molecules to be attached. Typically, the longer
the DNA molecules to be sequenced, the less density is needed. For
example, a 300 nucleotide long DNA is about 100 nm, thus ideally
there should be an area with a radius of greater than 100 nm with a
DNA molecule in the center. DNA can be immobilized in the region by
standard methods. For example, acrydite-modified DNA fragments can
be attached to a surface modified with thiol groups and
amine-modified DNA fragments can be attached to epoxy or aldehyde
modified surfaces.
[0034] Typical useful polymerase enzymes include DNA polymerases
with or without 3' to 5' exonuclease activities, such as for
example, E. coli DNA polymerase I, Klenow fragment of E. Coli DNA
polymerase I, phusion DNA polymerase, 9 N and Therminator DNA
polymerase, reverse transcriptase, Taq DNA polymerase, Vent DNA
polymerase (all available from New England Biolabs, Inc., Beverly,
Mass.), T4 and T7 DNA polymerases, and Sequenase (all available
from USB, Cleveland, Ohio). Nuclease-resistant nucleotides can be
ribonucleotides or other modified nucleotides. A variety of
polymerases are available that can incorporate ribonucleotides or
modified nucleotides into DNA, such as for example, the
commercially available Therminator DNA polymerase (available from
New England Biolabs, Inc., Beverly, Mass.) or genetically
engineered DNA polymerase. See also, for example, DeLucia, A. M.,
Grindley, N. D. F., Joyce, C. M., Nucleic Acids Research, 31:14,
4129-4137 (2003); and Gao, G., Orlova, M., Georgiadis, M. M.,
Hendrickson, W. A., Goff, S. P., Proceedings of the National
Academy of Sciences, 94, 407-411 (1997). Exemplary nuclease
resistant nucleotides that can be incorporated into growing DNA
strands but that are resistant to digestion by exonucleases (such
as the 3' to 5' exonuclease active DNA polymerases or exonuclease I
and III) include alpha-phosphorothioate nucleotides (available from
Trilink Biotechnologies, Inc., San Diego, Calif.). Additionally,
ribonucleotides can be incorporated into a growing DNA strand by
Therminator DNA polymerase or other genetically engineered or
mutated polymerases. Phi-29 DNA polymerase (available from New
England Biolabs) provides strand displacement activity and terminal
deoxynucleotide transferase provides template independent 3'
terminal base addition.
[0035] FIG. 8 provides a diagram of an exemplary DNA sequencing
system. In FIG. 8, a light source 605 is placed below a reaction
substrate 610 and an imager 615 is placed above the reaction
substrate 610. The reaction substrate 610 consists of a waveguide
and surface-attached DNA molecules to be sequenced and
surface-attached PPi chelating molecules (not shown). The light
source 605 is, for example, a 488 nm laser, a 514 nm laser, a 532
nm laser, and other light sources based on dyes. These light
sources are commercially available. The imager 615 is, for example,
a CCD (charge coupled device) camera, a cooled CCD camera, a deep
cooled EMCCD (electron multiplying charge coupled device) camera or
PMT (photomultiplier tube) array. Imaging devices are commercially
available from, for example, Hamamatsu Photonics, Hamamatsu City,
Japan. A light source control 620 controls the operation of the
light source 605 and is operably connected to the computer 625. The
light source control 620 is for controlling the intensity and
duration of light. An acousto-optic modulator can be used for this
purpose. Acousto-optic modulators are commercially available, for
example, from Sintec Optronics Pte Ltd., Singapore, Malaysia. The
imaging control 630 is operably coupled to the imager 615 and the
computer 625. Normally the high end cameras come with their own
controllers. However, there are some commercial available universal
controllers for image acquisition applications, such as NI image
acquisition cards (commercially available from, for example,
National Instruments Corporation, Austin, Tex.). Additionally, a
reagent storage and fluidic control device 635 provides reagents to
the reaction region and is operably coupled to the computer 625
that directs its operation. The reagents are typically supplied in
volumes in the .mu.l to ml range. Standard devices, such as
commonly used labware, plastic or glass tubes, or bottles can be
used to supply reagents for DNA sequencing reactions. Reagent
delivery in principle is similar to reagent delivery in HPLC (high
pressure liquid chromatography) applications. Various commonly used
pumps or vacuum device can be used. Typically, there are three
major parts in the reagent delivery system: a) reagent storage
devices, b) a reaction chamber, and c) waste container(s). A
fluidic pumping system under computer control (similar to a HPLC
system) is used to connect the three parts. The connection can be
done by tubing, or parts mechanically fabricated by well-known
methods. A mixing mechanism for solutions may also be used. Stored
reagents kept separately include, four solutions of signaling
nucleotides (fluorescently-labeled, each of the four nucleotides),
one to four solutions for bifunctional nucleotides (nuclease
resistant and 3' reversibly blocked dNTPs), an enzyme solution, a
washing solution which has typically the same composition as the
enzymatic reaction buffer, and a nucleotide de-blocking reagent.
Other reagent storage spaces may also provided for system
flexibility. Under the control of the computer program, one or more
reagents can be delivered to the reaction chamber. The reagents can
mixed before or after entering the chamber. Used reagents are
withdrawn and disposed in a waste container. These storage devices
may be stored at room temperature, or at 4.degree. C. A wash
solution containing the same buffer components as a reaction buffer
can be used to clean the surface of the waveguide. To reuse the
chelator, bound PPi can be removed using weak acid, such as acetic
acid (having a concentration, for example, of 1 mM to 1 M). The
waveguide surface can be reconditioned with the wash conditions. An
additional wash solution may be needed when additional components
required by the chelator need to be added back to the surface, such
as metal ions (Zn.sup.2+). Optionally, the device of FIG. 8 can be
a miniaturized device, such as a microfluidic or a nanofluidic
device. The computer automates the control of the delivery of
reagents, monitors the results from optical measurements, and
assembles sequence data from multiple reactions. Microscale fluidic
devices typically have interior features for fluid flow and
containment having diameters of 500 .mu.m or less. A micrometer
(.mu.m) is 10.sup.-6 meters. Nanoscale fluidic devices typically
have interior features for fluid flow and containment having
diameters of 500 nm or less. A nanometer (nm) is 10.sup.-9
meters.
[0036] In various embodiments of the invention, sequencing
substrates may be incorporated into a larger apparatus and/or
system. In certain embodiments, the substrate may be incorporated
into a micro-electro-mechanical system (MEMS). MEMS are integrated
systems comprising mechanical elements, sensors, actuators, and
electronics. All of those components may be manufactured by known
microfabrication techniques on a common chip, comprising a
silicon-based or equivalent substrate (See for example, Voldman et
al., Ann. Rev. Biomed. Eng., 1:401-425 (1999).) The sensor
components of MEMS may be used to measure mechanical, thermal,
biological, chemical, optical and/or magnetic phenomena. The
electronics may process the information from the sensors and
control actuator components such as pumps, valves, heaters,
coolers, and filters, thereby controlling the function of the
MEMS.
[0037] The electronic components of MEMS may be fabricated using
integrated circuit (IC) processes (for example, CMOS (complementary
metal-oxide semiconductor) and bipolar, or BICMOS processes). The
components may be patterned using photolithographic and etching
methods known for computer chip manufacture. The micromechanical
components may be fabricated using compatible micromachining
processes that selectively etch away parts of the silicon wafer or
add new structural layers to form the mechanical and/or
electromechanical components.
[0038] Basic techniques in chip manufacture include depositing thin
films of material on a substrate, applying a patterned mask on top
of the films by photolithographic imaging or other known
lithographic methods, and selectively etching the films. A thin
film may have a thickness in the range of a few nanometers to 100
micrometers. Deposition techniques of use may include chemical
procedures such as chemical vapor deposition (CVD),
electrodeposition, epitaxy and thermal oxidation and physical
procedures like physical vapor deposition (PVD) and casting.
[0039] In some embodiments of the invention, substrates may be
connected to various fluid filled compartments, such as
microfluidic channels, nanochannels, and or microchannels. These
and other components of the apparatus may be formed as a single
unit, for example in the form of a chip, such as semiconductor
chips and or microcapillary or microfluidic chips. Alternatively,
the substrates may be removed from a silicon wafer and attached to
other components of an apparatus. Any materials known for use in
such chips may be used in the disclosed apparatus, including
silicon, silicon dioxide, silicon nitride, polydimethyl siloxane
(PDMS), polymethylmethacrylate (PMMA), plastic, glass, and quartz.
These materials are especially useful for plasmonic waveguide
devices which do not require a substrate that is transparent. For
zero mode waveguide devices, the substrate itself or a layer of the
substrate typically is transparent, and a substance such as a glass
or silicon nitride is useful as the substrate material or as a
layer on a substrate in contact with the patterned metal layer.
EXAMPLES
[0040] Synthesis and attachment of pyrophosphate chelators to a
substrate surface: The pyrophosphate chelator was designed with
three main components: a binding site, a linker, and a handle. The
binding site was designed to bind PPi selectively, the linker
between the binding site and chelator provides steric flexibility
to the overall molecule if needed and the handle ensures that the
chelator can be attached to a surface. The selected PPi chelator
has demonstrated high binding capability to PPi. Referring to FIG.
9, the starting material for the synthesis of the
surface-attachable pyrophosphate chelator was
5-nitro-1,3-bishydroxymethylbenzene, whose hydroxyl groups were
tosylated to accelerate the substitution reaction in the next
synthesis reaction. The tosylate groups were replaced by
dipyridinylamine. The nitro group was reduced efficiently to an
amine group by catalytic hydrogenation using Pd C and H.sub.2. The
zinc nitrate (Zn(NO.sub.3).sub.2) was added afterwards to yield the
final functional pyrophosphate chelator.
[0041] The synthesized pyrophosphate chelator was immobilized on a
substrate surface that had been silanated. An aldehyde group was
used to functionalize the silicon surface through derivatization of
the silicon surface with 4-(triethoxysilyl)butyraldehyde. Reductive
amination with sodium triacetoxyborohydride was used to covalently
attach the pyrophosphate chelator to the derivatized substrate
surface. Immobilization of the pyrophosphate chelator was
confirmed: the substrate surface was characterized by ellipsometry,
atomic force microscope (AFM) and TOF-SIMS (time-of-flight
secondary ion mass spectroscopy). Monolayer thicknesses and sample
topography were consistent with step-by-step surface modification
of silicon substrate surface. Ellipsometry and AFM data indicated a
thickness of about 35 .ANG. for the pyrophosphate chelator and its
linker, consistent with the expected value. TOF-SIMS measurements
of modified substrate surfaces yielded the expected mass of the
immobilized pyrophosphate chelator while the pyrophosphate chelator
was not detected on several types of control samples.
[0042] Binding kinetics: The newly synthesized immobilizable
pyrophosphate chelator was subjected to selective binding studies
using a coumarin-based fluorescent dye,
(6,7-dihydroxy-2-oxo-2H-chromen-4-yl)methanesulfonate, and a
colorimetric dye, pyrocatechol violet (PV). In case of fluorescent
dye, binding to the chelator caused quenching of its fluorescence.
As more chelator was added, fluorescence intensity decreases
showing dose response as expected that reached a plateau near 10
.mu.M. The dose response curve was used to estimate the binding
constant for this fluorescent dye at 1.7.times.10.sup.6 M.sup.-1.
This binding constant was similar to what was reported for a
similar pyrophosphate chelator. When the colorimetric dye was used,
the binding to pyrophosphate chelator caused a detectable color
change from blue (free dye, .lamda..sub.max 444 nm) to yellow
(complex, .lamda..sub.max 624 nm). The peak absorption change from
blue (free dye, .lamda..sub.max 444 nm) to yellow (.lamda..sub.max
624 nm) indicated formation of chelator-dye complex. This color
change was visible to naked eye.
[0043] To study selectivity of the immobilizable pyrophosphate
chelator, the binding of PPi to the chelator was compared to the
binding of phosphate (Pi) and dATP. Both fluorescence and
absorption data indicated that the chelator showed selectivity for
PPi over Pi and dATP. A competitive displacement assay of the
immobilizable chelator with PPi, dATP, and Pi was performed. 1:1
mixtures of chelator and fluorescent dye were treated with various
concentrations of binders. Fluorescence was monitored at 480 nm
with excitation at 347 nm. Other dNTPs were also studied in
competitive displacement assays. The immobilizable chelator was
found to bind PPi preferentially over other dNTPs, similar to the
results for dATP.
* * * * *